This invention is concerned with the problem of producing hapten-specific antibodies when the haptan is inherently unstable, as with PGH.sub.2 and PGX, or when the hapten is changed during antibody production, as with certain PGE-type compounds. See, for example, Raz, et al., Eur. J. Biochem., 53, 145-150 (1975).
The specificity of the immune response is ancient knowledge; e.g., it was observed over the ages that persons who contracted smallpox, measles, etc. would not be reafflicted with the same disease but were not thereby protected from other diseases. The cross-reactivity of the immune response is also well known; e.g., the smallpox vaccine is derived from cowpox, not smallpox. The mechanism which creates antibodies which, although highly specific, may nevertheless cross-react with compounds other than the antigen which produced them is not fully understood. However, it is known that using substitutes which are merely gross space filling will not necessarily produce the desired antibodies. Thus, replacing the oxygen at C-9 in PGE.sub.1 with chlorine yields a compound of the formula ##STR1## which does not produce antibodies specific against PGE.sub.1 developing an RIA for prostaglandins, Cornette, et al., Radioimmunoassay of Prostaglandins, The Prostaglandins, 243-255, Futura Pub. Co., N.Y., (1972), found little cross-reactivity between prostaglandins with different ring structures: " . . . the specificity of the antibody is primarily determined by substituents on the cyclopentane ring . . . " (p. 247). See also: Salmon, Ng, and Karim, Prostaglandins, 9, 339-346 (1975).
Attempts have been made by different research groups to produce antibodies against PGE.sub.1. The results of cross reaction experiments are summarized in Table I. In each example, rabbits were inoculated with a PGE.sub.1 conjugate. The antibodies produced varied in selectivity. Relatively selective antibodies were produced in Example 1. Cross reaction with PGE.sub.2 was only 20%. Moderately selective antibodies were produced in Examples 3, 5, and 7. Cross reaction with PGE.sub.2 was about 50%. Non-selective antibodies were produced in Example 4. Cross reaction with E.sub.2 or A and B series prostaglandins was 100%. In certain extreme cases, antibodies produced against PGE.sub.1 conjugates showed greater selectivity to the B prostaglandins than to PGE.sub.1 (Example 2), and in another example (6), the A series prostaglandins were also recognized preferentially even though a PGE.sub.1 conjugate was used.
Similar results are seen in Table II from attempts to generate antibodies specific for PGE.sub.2. The antibodies varied in selectivity. Relatively selective antibodies were obtained in Examples 1 and 4, where only PGE.sub.1 cross-reacted. In Example 2 an E-specific antibody was produced but there was no selectivity between E.sub.1 and E.sub.2. Again, extreme cases were observed as in Example 5, were prostaglandins A.sub.2 and B.sub.2 were recognized preferentially even though an E.sub.2 conjugate was used. In Example 3, the A prostaglandins were recognized preferentially, although an E.sub.2 conjugate was used.
Thus, cross-reaction is likely to be varied and unpredictable for some PGE-type compounds.
Table I __________________________________________________________________________ % Cross Reaction Example Conjugate PGE.sub.1 PGE.sub.2 PGA.sub.1 PGA.sub.2 PGB.sub.1 PGB.sub.2 __________________________________________________________________________ 1 PGE.sub.1 -thyroglobulin (1) 100 20 -- -- -- -- 2 PGE.sub.1 -poly-l-lysine (2) 100 10 220 440 31,000 8,500 3 PGE.sub.1 -porcine gamma globulin (3) 100 50 -- -- 10 -- 4 PGE.sub.1 -BSA (4) 100 100 100 100 -- 100 5 PGE.sub.1 -BSA (4) 100 40 25 10 -- -- 6 PGE.sub.1 -BSA (4) 100 90 10,000 1000 100,000 50,000 7 PGE.sub.1 -BSA (5) 100 20 6 -- 66 -- __________________________________________________________________________ References: (1) Ritzi and Stylos, Prostaglandins, 8, 55-66 (2) Levine et al., J. Biol. Chem., 246, 6782-6785 (1974) (3) Jubiz et al., Prostaglandins, 2, 471-489 (1972) (4) Yu and Burke, Prostaglandins, 2, 11-22 (1972) (5) Maclouf et al., FEBS Letters, 56, 273-278 (1975)
Table II __________________________________________________________________________ % Cross Reaction Example Conjugate PGE.sub.2 PGE.sub.1 PGA.sub.2 PGA.sub.1 PGB.sub.2 PGB.sub.3 __________________________________________________________________________ 1 PGE.sub.2 -BSA (1) 100 10 7 7 7 7 2 PGE.sub.2 -KLH (2) 100 110 -- -- -- -- 3 PGE.sub.2 -BSA (3) 100 73 400 200 -- 62 4 PGE.sub.2 -hen gamma globulin (4) 100 15 3 -- -- -- 5 PGE.sub.2 -BSA (5) 100 55 550 -- 580 -- __________________________________________________________________________ References: (1) Bauminger et al., Prostaglandins, 4, 313-324 (1973) (2) Ritzi and Stylos Prostaglandins, 8, 55-66 (1974) (3) Zusman et al., Prostaglandins, 2, 41-53 (1972) (4) Christensen and Leyssac, Prostaglandins, 11, 399-420 (1976) (5) Raz et al., Eur. J. Biochem., 53, 145-150 (1975)
Performing radioimmunoassays (RIAs) for various of the PG-type compounds has been problematical primarily because of their instability. For example, PGE.sub.2 dehydrates into PGA.sub.2, which is further isomerized to PGB.sub.2 during antibody production. The antibodies produced cross-react with PGA.sub.2 and PGB.sub.2 thereby interfering with the RIA for PGE.sub.2.
As used herein, the term "prostaglandin" (or "PG") refers to those cyclopentane-containing carboxylic acids derived from mammalian tissues which are structural derivatives of prostanoic acid: ##STR2## See Bergstrom, et al. Pharmacol. Rev. 20, 1 (1968) and references cited therein. For example, prostalandin E.sub.2 (PGE.sub.2) has the following structure: ##STR3##
PGH.sub.2 has the following structure: ##STR4##
PGX (also known as prostacyclin, PGl.sub.2, and 9-deoxy-6,9.alpha.-epoxy-.DELTA..sup.5 -PGF.sub.1 .alpha.) has the following structure: ##STR5## See also Nelson, Prostaglandin Nomenclature, J. of Med. Chem., 17, 911 (1974).
The term "prostaglandin analog" herein refers to those compounds structurally related to the prostaglandins (in that they exhibit a cyclopentane ring and a pair of side chains attached to adjacent carbon atoms of the ring) which retain certain characteristic biological properties of the prostaglandins. See Bergstrom, cited above. Various structural modifications of the prostaglandins are known to produce useful prostaglandin analogs. For example, the replacement of the carboxy with a hydroxymethyl is known, substitution of a methyl, ethyl, or fluoro for a hydrogen at, for example, C-16 is known. Further, partially deoxygenated prostaglandins are known to be useful prostaglandin analogs. Accordingly, 9-deoxy prostaglandins are known. Finally, there are known prostaglandin analogs wherein the double bonds of, for example, PGF.sub.2 .alpha. are shifted, e.g., cis-4,5-didehydro-PGF.sub.1 .alpha., or replaced by triple bonds, e.g., 13,14-didehydro-PGF.sub.2 .alpha..
As used herein, the terms "PGE.sub.1 -type" and "PGE.sub.2 -type" compound refer to PGE.sub.1, PGE.sub.2, or their respective analogs of the formula ##STR6## wherein M.sub.1 is ##STR7## wherein R.sub.5 is hydrogen or methyl; wherein L.sub.1 is ##STR8## or a mixture of ##STR9## and ##STR10## wherein R.sub.3 and R.sub.4 are hydrogen, methyl, or fluoro, being the same or different, with the proviso that one of R.sub.3 and R.sub.4 is fluoro only when the other is hydrogen or fluoro;
wherein Z.sub.1 is
(1) cis--CH.dbd.CH--CH.sub.2 --(CH.sub.2).sub.g --CH.sub.2 --, PA0 (2) cis--CH.dbd.CH--CH.sub.2 --(CH.sub.2).sub.g --CF.sub.2 --, PA0 (3) cis--CH.sub.2 --CH.dbd.CH--(CH.sub.2).sub.g --CH.sub.2 --, PA0 (4) --(CH.sub.2).sub.3 --(CH.sub.2).sub.g --CH.sub.2 --, PA0 (5) --(CH.sub.2).sub.3 --(CH.sub.2).sub.g --CF.sub.2 --, PA0 (6) --CH.sub.2 --O--CH.sub.2 --(CH.sub.2).sub.g --CH.sub.2 --, PA0 (7) --C.tbd.C--CH.sub.2 --(CH.sub.2).sub.g --CH.sub.2 --, PA0 (8) --CH.sub.2 --C.tbd.C--(CH.sub.2).sub.g --CH.sub.2 --, ##STR11## wherein g is one, 2, or 3; PA0 (1) --(CH.sub.2).sub.m --CH.sub.3, ##STR12## wherein m is one to 5, inclusive; n is zero or one; T is chloro, fluoro, trifluoromethyl, alkyl of one to 3 carbon atoms, inclusive, or alkoxy of one to 3 carbon atoms, inclusive; and s is zero, one, 2, or 3, the various T's being the same or different, with the proviso that not more than two T's are other than alkyl, with the further proviso that R.sub.7 is ##STR13## wherein T and s are as defined above, only when R.sub.3 and R.sub.4 are hydrogen or methyl, being the same or different; and
wherein R.sub.7 is
wherein X.sub.1 is --COOR.sub.1 wherein R.sub.1 is hydrogen or a pharmacologically acceptable cation.
"PGE-type" consists of PGE.sub.1 -type and PGE.sub.2 -type.
The terms "prostaglandin mimic" and "authentic prostaglandin" are used herein to differentiate between certain stable analogs and their corresponding prostaglandin-type compounds. These stable analogs (mimics) are characterized in that the antibodies formed against the analogs are highly cross-reactive (&gt;30%) with the authentic prostaglandins. Specifically, the mimics to PGE.sub.1 - and PGE.sub.2 -type compounds are 9-deoxy-9-methylene PGF-type compounds having the ring structure ##STR14## where the side chains are the same as shown in formula I. The mimic to PGH.sub.2 is (5Z, 9.alpha., 11.alpha., 13E, 15S)-9,11-azo-15-hydroxyprosta-5,13-dien-1-oic acid: ##STR15## The mimic to PGX is 9-deoxy-6,9.alpha.-epoxy-PGF.sub.1 .alpha.: ##STR16##
PGE-type compounds are known to be useful pharmacological agents capable to conventional formulations and administration by a wide variety of routes. See U.S. Pat. No. 3,903,297 for a description of typical methods of formulation and administration. See also Bergstrom, et al., cited above.
PGH.sub.2, derived endogenously from arachidonic acid, is a transient regulator in mammalian cells. (See Hamberg and Samuelsson, Proc. Natn. Acad. Sci., U.S.A. 70, 889-903 (1973).) Upon formation, the endoperoxide is rapidly converted to PGF.sub.2 .alpha., PGE.sub.2, their 15-keto-PG metabolites, and thromoboxane A.sub.2. (See Hamberg, et al., Proc. Natn. Acad. Sci., U.S.A. 72, 2994-2998 (1975) and Monocada, et al., Nature 263, 663-665 (1976).) The enzymatic composition of each tissue governs the nature and extent of these conversions. By its direct influence and as precursor for other PGs. PGH.sub.2 is a primary cellular mediator; unfortunately, its short half-life makes direct determination of PGH.sub.2 -related phenomena difficult.
PGX is also endogenously formed from arachidonic acid. Of physiological significance is its potent opposition to the platelet aggregating properties of PGH.sub.2 and thromboxane A.sub.2, both of which have been implicated in the occurrence of thrombosis. (See Horton, Nature, 263, 637, (1976).) Like PGH.sub.2, PGX is intrinsically unstable.
Radioimmunoassay (RIA) is a sensitive, analytical technique in which a compound to be measured and the same compound is a radioactive form compete for binding sites on antibodies showing a selective affinity for the compound. The discovery of the techniques as it is used today is generally credited to Berson and Yalow, J. Clin. Invest., 38, 1996, (1959). The principles of radioimmunoassay determination have been described by Hawker, Radioimmunoassay and Related Methods, Anal. Chem., 45, 878A-888A, (1973), and Odell and Daughaday, Principles of Competitive Binding Assays, J. B. Lippincott, Phil., (1971). The method has been applied to the assay of numerous compounds.
A useful RIA method may be developed only when one has:
(1) The radiolabeled form of the compound to be measured PA1 (2) Antibodies with the ability to bind the compound to be measured.
Ordinarily, the procurement of the radiolabeled form of the compound to be measured precedes the attempt to produce antibodies. The limiting step in the development of a radioimmunoassay method then is the successful production of antibodies with the ability to bind the compound to be measured.
Antibodies are plasma proteins synthesized in humoral immune responses which are capable of combining with the provoking antigens. There are several types of plasma proteins with associated antibody activity and these are denoted collectively as immunoglobulins. Antiserum is serum which contains antibodies.
Antigens (immunogens) are substances capable of provoking an immune response of any type in an immunologically competent vertebrate. Antigens are substances of high molecular weight (&gt;2000), usually proteins or carbohydrates.
Haptens are incomplete antigens; i.e., substances by themselves incapable of provoking an immune response, but able to serve as partial immunogens when bound to another substance denoted as a carrier molecule. Haptens are usually of low molecular weight (&lt;500) and may be of relatively simple structure. (See Sela (ed.). The Antigens, Academic Press, N.Y., 1973).
Vertebrates stimulated (inoculated) by a conjugate (hapten-carrier molecule complex) will produce antibodies capable of reacting with the conjugate, the carrier molecule alone, and the hapten alone.